1. Field of the Invention
The present invention relates to polyprotein-producing viral vectors for production of non-native proteins and which employ proteolytic digestion of the non-native proteins by the viral vector's own proteases.
2. Description of the Prior Art
Recombinant DNA techniques and genetic technologies have led to progress in the development of gene transfer into organisms, both plant and animal. Investigators have attempted to produce pharmaceuticals, chemicals, and biologicals by gene transfer techniques. Even though a gene may have been identified, cloned, and engineered, it is still necessary to introduce the gene into a host cell in which the gene may be expressed.
Foreign genes may be expressed in the host from DNA integrated into the host genome (transgenic organism), or from extrachromosomal pieces of DNA or RNA which are not inherited (through transient expression vectors). A variety of methods have been developed and are currently available for the transformation of various cells with genetic material. The most success has been achieved with dicotyledonous plants with some limited success reported for the monocotyledonous plants. The most highly developed system for higher plants transformation is derived from the tumor-inducing mechanism of the soil bacterium Agrobacterium tumefaciens. The Agrobacterium/Ti plasmid system exploits the elements of the Agrobacterium transformation mechanism. Alternative methods for delivering genetic material to plants include the direct incorporation of DNA such as the transformation of plant cell protoplast. Other techniques include microinjection or DNA bombardment.
Transient expression vectors can be introduced directly into the recipient cell, and result in gene expression in a short period of time, generally 24-48 hours. Viral vectors (both DNA and RNA) offer possibilities as plant and animal transformation vectors. Viral vectors have some advantages over vectors involving non-viral DNA in certain circumstances. Advantages of viral vectors include improvements in the ease of gene introduction.
Viruses that have been shown to be useful for the transformation of plant hosts include caulimovirus (CaMV), tobacco mosaic virus (TMV) and brome mosaic virus (BMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BMV), EPA 278,667 (BV), Brisson, N. et al., Met. Enzymol. 118:659 (1986) (CaMV), Guzman et al. Commun. Molec. Biol.: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172- 189 (1988), and WO 93/03161 (TMV). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261.
For RNA viruses, a primary consideration is designing a vector that produces infectious transcripts, and therefore preserves the terminal structure of the RNA in order for the transcript to be a good substrate for the viral replicase. Bacteriophage promoters have proven ideal for directing the synthesis of RNA from DNA clones. One RNA virus expression vector, pPM1, utilized a modified lambda phage P.sub.r promoter. This vector was used to synthesize infectious transcripts of brome mosaic virus (BMV) genomic RNA from BMV cDNA clones. However, most of these viral vectors have not been capable of systemic spread in the plant and expression of the non-viral foreign genes in the majority of the plant cells in the whole plant. Another disadvantage of many of the prior art viral vectors is that they are not stable for the maintenance of non-viral foreign genes. The use of RNA vectors has also been limited by the instability of inserted foreign sequences and disruption of systemic virus spread after replacement of virus genes involved in movement. TMV viral vectors require a subgenomic promoter for operation. The prior art viral vectors do not provide a means for using viral proteases to cleave the foreign proteins made by the transformed plants. Consequently, there is a need for vectors which enable enhanced systemic expression of a nucleic acid sequence after a short inoculation time and for vectors which can be used to produce both native and non-native proteins relative to the host cell, particularly plant cells, while allowing cleavage of the produced protein by viral proteases. cl SUMMARY OF THE INVENTION
It is an object of the invention to provide a polyprotein-producing virus expression vector adapted to encoding at least one sequence for at least one heterologous protein. The vector may be used to express heterologous proteins in plants or plant cells.
Accordingly, the present invention provides a viral expression vector encoding for at least one protein non-native to the vector that is released from at least one polyprotein expressed by said vector by proteolytic processing catalyzed by at least one protease in said polyprotein, said vector comprising: (a) at least one promoter; (b) cDNA, wherein said cDNA comprises a cDNA sequence which codes for at least one polyprotein from a polyprotein-producing virus; (c) at least one unique restriction site flanking a 3' terminus of said cDNA; and (d) a cloning vehicle.
The present invention also provides a viral expression vector encoding for at least one protein non-native to the vector that is released from at least one polyprotein expressed by said vector by proteolytic processing catalyzed by at least one protease in said polyprotein, said vector comprising: (a) at least one promoter; (b) cDNA, wherein said cDNA comprises a cDNA sequence which codes for at least one polyprotein from a polyprotein-producing virus, and wherein said polyprotein comprises at least one protein non-native to the vector; (c) at least one unique restriction site flanking a 3' terminus of said cDNA; and (d) a cloning vehicle.
The present invention also provides a method for producing an expression vector encoding for at least one selected protein non-native to the vector that is released from at least one polyprotein expressed by said vector by proteolytic processing catalyzed by proteases in said polyprotein, said method comprising: (a) reverse transcribing a polyprotein-producing RNA into cDNA; (b) introducing at least one unique restriction site flanking a 3' terminus of said cDNA; and (c) inserting said cDNA into a cloning vehicle.
The present invention also provides a method for producing an expression vector, adapted for expressing in a host cell at least one selected protein non-native to the vector that can be released from at least one polyprotein by proteolytic processing catalyzed by proteases in said polyprotein, said method comprising: (a) reverse transcribing a polyprotein-producing RNA into a first cDNA; (b) introducing at least one unique restriction site flanking a 3' terminus of said first cDNA; and (c) inserting into said first cDNA a second cDNA sequence wherein said second cDNA sequence encodes a protein non-native to the vector; and (d) inserting said first and second cDNA into a cloning vehicle.
The present invention also provides a method for expressing at least one protein in a host cell, wherein the protein is non-native to the host cell, said method comprising infecting a host cell susceptible to a polyprotein-producing virus with an expression vector, wherein said vector comprises a cDNA sequence encoding at least one polyprotein, each of said polyprotein comprising at least one protein non-native to the vector and at least one protease, and wherein said protein non-native to the vector is released from said polyprotein by proteolytic processing catalyzed by said protease encoded by said cDNA, and expressing said protein non-native to the vector in said host cell.
These and other advantages of the present invention will become apparent from the following detailed description.